Structure and Function of Sodium-Calcium Exchangers

钠钙交换体的结构和功能

基本信息

批准号：
8841747
负责人：
Dinesh A Yernool
金额：
$ 28.67万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-15 至 2017-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8841747
关键词：
Active Biological Transport Architecture Binding Sites Biochemical Calcium Calcium ion Cardiac Cardiac Muscle Contraction Cardiac Myocytes Cations Cell Death Cell division Cell membrane Cell physiology Cells Chimeric Proteins Communities Complex Crystallization Data Development Disease Drug Design Elements Family Fertilization Foundations Future Genetic study Goals Health Heart Diseases Homeostasis Homologous Gene Ii-Key Immunoglobulin Fragments Integral Membrane Protein Ion Channel Ion Transport Kinetics Knowledge Laboratories Lead Life Liposomes Membrane Membrane Proteins Membrane Transport Proteins Methodology Methods Mission Modeling Molecular Molecular Genetics Myocardial Ischemia Myocardial dysfunction Neurons Outcome Physiological Property Protein Family Proteins Proteolysis Public Health Pump Recurrence Regulation Research Resolution Signal Transduction Sodium-Calcium Exchanger Specificity Structural Models Structure Structure-Activity Relationship Study models Time Toxic effect Transmembrane Domain United States National Institutes of Health X-Ray Crystallography base biophysical properties cost design experience improved inhibitor/antagonist neuron loss neurotransmission novel novel strategies optical imaging overexpression receptor reconstitution small molecule structural biology three dimensional structure three-dimensional modeling transport inhibitor

项目摘要

DESCRIPTION (provided by applicant): Flux of calcium ions, potent signals, controls many if not all cellular processes from fertilization and cell division to eventual cell death. Sodium/calcium exchangers (NCX), high-capacity integral membrane transporters, are the principal Ca2+ pumps in the plasma membranes of most cells. The unmatched ability of NCX to rapidly extrude Ca2+ using preexisting Na+-gradient potentials is critical to the normal function of excitable cells such as cardiac myocytes and neurons. These cells undergo quick, recurrent oscillations in levels of intracellular Ca2+ to achieve cardiac muscle contraction and neuronal signaling, respectively; and perturbations in NCX activity have been implicated in cardiac dysfunction and neuronal cell death. At present, an atomic-resolution structure of NCX has not been determined. Lack of 3D structure impedes the elucidation of structure-function relationships that are key to understanding NCX's mechanism of action. Our long-term goal is to describe at the atomic level the mechanism of transport and regulation of Ca2+/cation exchange in cardiac and neuronal cells. Toward this goal, the specific aims proposed here are as follows: 1. Heterologous overexpression of membrane proteins using a novel method. Optimized expression methods developed in my laboratory to increase membrane protein yields and reduce their host toxicity will enable us to produce enough protein for biochemical, biophysical, and crystallographic analyses. Initial expression targets will be prokaryotic NCX homologs selected for their significant sequence identity to eukaryotic sodium-calcium exchangers. Subsequently we will demonstrate our method's applicability to a large panel of membrane protein targets and investigate the molecular basis of overexpression toxicity. 2. Demonstration that prokaryotic NCX is an appropriate model for studies of Na+/Ca2+ exchange. This will be achieved by determining the oligomerization state and topology of the transporter, followed by characterization of structure-function and transport properties of prokaryotic NCX homologs by reconstitution into liposomes. Wild-type ion transport kinetics will be determined and inhibitors of transport identified. 3. Structure. We will crystallize a prokaryotic NCX homolog(s) alone and/or in complex with antibody fragments to determine and analyze structure(s). Combining biochemical and biophysical data with the crystal structure of an NCX homolog will lead to development of structure-based mechanistic models for Na+-Ca2+ exchange, spur new structure-based hypotheses to understand the mechanism of cardiac NCX, and may inspire structure-based drug design to find pharmacologically active agents.

描述（由申请人提供）：钙离子的通量，有效的信号，控制许多（如果不是全部）从受精和细胞分裂到最终细胞死亡的细胞过程。钠/钙交换器（NCX），高容量积分膜转运蛋白，是大多数细胞质膜中的主要Ca2+泵。 NCX使用先前存在的Na+差异电位快速挤出Ca2+的能力对于可激发细胞（例如心肌细胞和神经元）的正常功能至关重要。这些细胞在细胞内Ca2+水平上进行快速，复发的振荡，分别获得心肌收缩和神经元信号传导。 NCX活性的扰动与心脏功能障碍和神经元细胞死亡有关。目前，原子分辨率 NCX的结构尚未确定。缺乏3D结构阻碍了结构 - 功能关系的阐明，这是理解NCX的作用机理的关键。我们的长期目标是在原子水平上描述心脏和神经元细胞中Ca2+/阳离子交换的运输和调节机制。为了实现这一目标，这里提出的特定目的如下：1。使用一种新方法对膜蛋白的异源过表达。在我的实验室开发的优化表达方法以增加膜蛋白的产量并降低其宿主毒性将使我们能够生产出足够的蛋白质来进行生化，生物物理和晶体学分析。最初的表达靶标将是原核NCX同源物，以供其与真核钠钙化器交换器相关的显着序列身份。随后，我们将证明我们的方法适用于大型膜蛋白靶标，并研究过表达毒性的分子基础。 2。证明核NCX是用于研究Na+/Ca2+交换的合适模型。这将通过确定转运蛋白的寡聚状态和拓扑结构来实现，然后通过重新结构为脂质体来表征结构功能和原核生物NCX同源物的运输特性。将确定野生型离子运输动力学，并确定运输的抑制剂。 3。结构。我们将单独结晶核NCX同源物和/或与抗体片段中的复合物来确定和分析结构。将生化和生物物理数据与NCX同源物的晶体结构相结合，将导致开发基于结构的机械模型，用于NA+ -CA2+交换，刺激了新的基于结构的假设了解心脏NCX的机制，并可能激发基于结构的药物设计药理学活性剂。